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Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reesei

Karlsson, Johan; Saloheimo, Markku; Siika-aho, Matti; Tenkanen, Maija; Penttilä, Merja and Tjerneld, Folke LU (2001) In European Journal of Biochemistry 268(24). p.6498-6507
Abstract
There are currently four proteins in family 61 of the glycoside hydrolases, from Trichoderma reesei, Agaricus bisporus, Cryptococcus neoformans and Neurospora crassa. The enzymatic activity of these proteins has not been studied thoroughly. We report here the homologous expression and purification of T. reesei Cel61A [previously named endoglucanase (EG) IV]. The enzyme was expressed in high amounts with a histidine tag on the C-terminus and purified by metal affinity chromatography. This is the first time that a histidine tag has been used as a purification aid in the T. reesei expression system. The enzyme activity was studied on a series of carbohydrate polymers. The only activity exhibited by Cel61A was an endoglucanase activity... (More)
There are currently four proteins in family 61 of the glycoside hydrolases, from Trichoderma reesei, Agaricus bisporus, Cryptococcus neoformans and Neurospora crassa. The enzymatic activity of these proteins has not been studied thoroughly. We report here the homologous expression and purification of T. reesei Cel61A [previously named endoglucanase (EG) IV]. The enzyme was expressed in high amounts with a histidine tag on the C-terminus and purified by metal affinity chromatography. This is the first time that a histidine tag has been used as a purification aid in the T. reesei expression system. The enzyme activity was studied on a series of carbohydrate polymers. The only activity exhibited by Cel61A was an endoglucanase activity observed on substrates containing β-1,4 glycosidic bonds, e.g. carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC) and β-glucan. The endoglucanase activity on CMC and β-glucan was determined by viscosity analysis, by measuring the production of reducing ends and by following the degradation of the polymer on a size exclusion chromatography system. The formation of soluble sugars by Cel61A from microcrystalline cellulose (Avicel; Merck), phosphoric acid swollen cellulose (PASC), and CMC were analysed on a HPLC system. Cel61A produced small amounts of oligosaccharides from these substrates. Furthermore, Cel61A showed activity against cellotetraose and cellopentaose. The activity of Cel61A was several orders of magnitude lower compared to Cel7B (previously EG I) of T. reesei on all substrates. One significant difference between Cel61A and Cel7B was that cellotriose was a poor substrate for Cel61A but was readily hydrolysed by Cel7B. The enzyme activity for Cel61A was further studied on a large number of carbohydrate substrates but the enzyme showed no activity towards any of these substrates. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Trichoderma reesei, endoglucanase, cellulase, histidine tag
in
European Journal of Biochemistry
volume
268
issue
24
pages
6498 - 6507
publisher
Wiley-Blackwell
external identifiers
  • scopus:0035663210
ISSN
0014-2956
DOI
10.1046/j.0014-2956.2001.02605.x
language
English
LU publication?
yes
id
759e7010-b956-4a72-a9f9-938a8ead2ed3 (old id 125018)
date added to LUP
2007-07-06 09:10:56
date last changed
2018-10-14 04:11:39
@article{759e7010-b956-4a72-a9f9-938a8ead2ed3,
  abstract     = {There are currently four proteins in family 61 of the glycoside hydrolases, from Trichoderma reesei, Agaricus bisporus, Cryptococcus neoformans and Neurospora crassa. The enzymatic activity of these proteins has not been studied thoroughly. We report here the homologous expression and purification of T. reesei Cel61A [previously named endoglucanase (EG) IV]. The enzyme was expressed in high amounts with a histidine tag on the C-terminus and purified by metal affinity chromatography. This is the first time that a histidine tag has been used as a purification aid in the T. reesei expression system. The enzyme activity was studied on a series of carbohydrate polymers. The only activity exhibited by Cel61A was an endoglucanase activity observed on substrates containing β-1,4 glycosidic bonds, e.g. carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC) and β-glucan. The endoglucanase activity on CMC and β-glucan was determined by viscosity analysis, by measuring the production of reducing ends and by following the degradation of the polymer on a size exclusion chromatography system. The formation of soluble sugars by Cel61A from microcrystalline cellulose (Avicel; Merck), phosphoric acid swollen cellulose (PASC), and CMC were analysed on a HPLC system. Cel61A produced small amounts of oligosaccharides from these substrates. Furthermore, Cel61A showed activity against cellotetraose and cellopentaose. The activity of Cel61A was several orders of magnitude lower compared to Cel7B (previously EG I) of T. reesei on all substrates. One significant difference between Cel61A and Cel7B was that cellotriose was a poor substrate for Cel61A but was readily hydrolysed by Cel7B. The enzyme activity for Cel61A was further studied on a large number of carbohydrate substrates but the enzyme showed no activity towards any of these substrates.},
  author       = {Karlsson, Johan and Saloheimo, Markku and Siika-aho, Matti and Tenkanen, Maija and Penttilä, Merja and Tjerneld, Folke},
  issn         = {0014-2956},
  keyword      = {Trichoderma reesei,endoglucanase,cellulase,histidine tag},
  language     = {eng},
  number       = {24},
  pages        = {6498--6507},
  publisher    = {Wiley-Blackwell},
  series       = {European Journal of Biochemistry},
  title        = {Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reesei},
  url          = {http://dx.doi.org/10.1046/j.0014-2956.2001.02605.x},
  volume       = {268},
  year         = {2001},
}